First report of Gnomoniopsis castaneae associated with branch dieback on chestnut tree (Castanea sativa) in Southern Chile.

European chestnut (Castanea sativa Mill.) currently reaches 1,470 ha, distributed from the Maule region to the Los Rios region in Chile. Almost 3000 tons of fruit have been exported in the last three years. A survey was carried out in January 2023 in an eight-year-old orchard located in Vilcún (38°34'46.22"S 72° 9'58.61"O), Araucanía Region. Chestnut trees with branch die back and reduced growth and vigor were detected. The incidence in the orchard was 3% (6 out of 200 trees) estimated by visual observation. Cross and longitudinal sections of the woody trunk of two trees were collected and examined, and an internal dark-brown discoloration to partial necrosis lesion was observed. To identify the causal agent, small pieces of wood from the edge of the symptomatic area were surface sterilized with 70% ethanol, rinsed twice with sterile distilled water, blotted on dry sterile filter paper, plated on potato dextrose agar (PDA) and incubated at 22°C. Fungal colonies were consistently isolated, and after 5 days, pure cultures were obtained by transferring mycelium to new PDA plates, preliminarily identified as Gnomoniopsis sp. (Visentin et al. 2012, Shuttleworth 2012). All cultures exhibited characteristics consistent with the description of G. castaneae (Syn. G. smithogilvyi), such as concentric development of greyish-brown mycelium, abundant stroma, hyaline conidia of 7.2 ±0.54 (6.1-8.1) X 2.3 ±0.26 (1.5-2.9) µm (n= 30), mainly biguttulate and fusoid. Total DNA was extracted, rDNA amplified using ITS1/ITS4 primers (White et al. 1990), and the fragment was Sanger sequenced and the sequence was deposited in GenBank (OR665735). BLAST analysis revealed a 99% identity to G. castaneae (MH384925). In addition, the DNA of the isolate was evaluated in a species-specific multiplex PCR (Silva-Campos et al. 2022), and the amplicons were electrophoretically separated, giving a similar band profile to G. smithogilvyi RGM 2903 and RGM 2904 strain from Chilean Collection of Microbial Genetic Resources. Pathogenicity of G. castaneae isolate (CV-11) was tested on ten replicates of 3-year-old C. sativa plants. Two wounds were made on the same season growing shoot and two on the previous season shoot. Longitudinal wounds (5 mm long, 4 mm wide and 2 mm depth) were made using a scalpel without removing the outer bark to inoculate the plants. Each wound was inoculated with a 5-mm mycelium plug, covered with the outer bark, and wrapped with Parafilm. Plugs of PDA were placed onto the wounds of two plants as control. The plants were kept in a growth chamber (22 ±1 0C and 90± 5% RH). All plants showed dark brown cankers measuring 20 to 40 mm long two weeks after inoculation. Also, most plants inoculated in the same season shoot presented wilted and chlorotic foliage. Mature conidiomata with cirri developed in most of the cankers. No symptoms were observed in the control. Fungal colonies of G. castaneae were reisolated on PDA from all inoculated chestnut plants and were not recovered from the controls. Recently, G. smithogilvyi has been identified as the causal agent of brown rot on chestnut nuts in Chile (Cisterna Oyarce et al. 2022); however, in several countries, it has also been associated as the causal agent of cankers in branch and stem of chestnut, as well as an endophyte in different hardwood species. Future studies on the incidence of this pathogen and its impact on chestnut yield should be carried out in the producing regions because it represents an emerging threat to Chilean chestnut production.

First Report of Beet Western Yellows Virus Infecting Vasconcellea x heilbornii (Caricaceae) in Ecuador.

Vasconcellea x heilbornii, known as babaco, is a hybrid native to Ecuador grown in small orchards in sub-tropical highland regions. Over the last decade, several viruses have been identified in babaco using high-throughput sequencing (HTS) (Cornejo-Franco et al. 2020, (Reyes-Proaño et al. 2023). In 2021, total RNA from a babaco plant showing distinctive leaf yellowing was extracted using the PureLink RNA Mini Kit (Thermo Fischer Scientific, USA) and subjected to HTS on an Illumina NovaSeq6000 system as 150 paired-end reads (Macrogen Inc., South Korea). Library construction was done using the TruSeq Stranded Total RNA Sample kit with Plant Ribo-Zero, as described (Villamor et al. 2022). Reads were processed using BBDuk and de novo assembled using SPAdes 3.15. both implemented in Geneious 2022. Contig analysis was done by BLASTx using the NCBI viral sequence database (as of November 2022). HTS generated 54 million reads, of which 12% assembled into contigs corresponding to genomes of previously reported babaco viruses including babaco virus Q (BabVQ), babaco nucleorhabdovirus 1 (BabRV1) and babaco ilarvirus 1 (BabIV1). Interestingly, 144 reads (0.0003%) assembled into seven contigs ranging from 100 to 480 nucleotides (nt) in length. These contigs showed homology, with 97% amino acid (aa) identity (100% query coverage), to regions of the RNA-dependent-RNA-polymerase (RdRp) of beet western yellows virus (BWYV, Acc. No. NC_004756), a member of the Polerovirus genus. To confirm the occurrence of BWYV in babaco, double-stranded RNA (dsRNA) was extracted from 15 g of leaf tissue from the original sample as described (Dodds et al. 1984) and used as template for reverse-transcription (RT)-PCR using overlapping primers designed to span all short contigs. RT-PCR amplified fragments were cloned into a pGEM®T-easy vector (Promega, USA) and sequenced by the Sanger method (Macrogen Inc., South Korea). The sequences were assembled into a single 2.7 kbp BWYV genome fragment comprising the complete protein 1 (P1) and partial RdRp gene (GenBank Acc. No. PP480670). Sequence alignments between the partially sequenced genome of the babaco isolate and its corresponding fragment from the closest BWYV isolate (NC_004756) revealed 94% and 97% identities at the nt and aa levels, respectively. To assess the prevalence of BWYV in babaco, 30 leaf samples showing yellowing symptoms from Pichincha (n=15) and Azuay (n=15) provinces were tested by RT-PCR using total RNA. Total RNA extraction and reverse transcription were done using the methodology described by Halgren et al. (2007). For RT-PCR, the primer set BWYV_Bab_F: 5'-CAGTGTCCTCCAAGTGCAACAT-3' / BWYV_Bab_R: 5'GGTTCCTTCCCAGTTTGGTGGT-3', which amplifies a 235 nt-long P1 region, was used. Three RT-PCR products from each positive sample were purified using the GeneJET PCR clean-up kit (Thermo Scientific, USA) and sequenced. BWYV was confirmed in 9 out of 15 samples (60%) from Pichincha, and in 10 out of 15 samples (64%) from Azuay. Samples were also tested for additional babaco viruses as described (Reyes-Proaño et al. 2023). All BWYV-infected plants turned out positive for papaya ringspot virus (PRSV), babaco mosaic virus (BabMV), BabVQ, and BabIV1. Hence, the impact of BWYV infection on babaco plants in single and mixed infections warrants further investigation. To the best of our knowledge, this is the first report of BWYV in a crop in Ecuador, and the first time it has been found in a Caricaceae species.

First report of Erwinia papayae associated with papaya mushy canker disease in Guam.

Carica papaya (papaya) in Guam, USA may experience soft rot symptoms, often referred to as mushy canker disease. Disease symptoms first appear as expanding water-soaked dark-green stem lesions or leaf spotting with chlorotic halos. Defoliation at petiole-stem junctions and crown necrosis leads to plant death. Papaya diseases caused by Erwinia spp. are documented in nearby tropic regions such as the Northern Mariana Islands (Trujillo and Schroth 1982), the Philippines (Dela Cueva et al. 2017), Japan (Hanagasaki et al. 2020), and Indonesia (Suharjo et al., 2021). The pathogen was isolated from symptomatic papaya stem sections (cv. Red Lady) from a nursery at the University of Guam Agriculture and Life Sciences building in April 2023. Approximately 20% of seedlings collapsed from stem soft rot, with nearly all plants showing varying degrees of water-soaked lesions on leaves or stems. Stem tissue from lesion margins was excised, surface sterilized with 70% EtOH, and macerated in sterile water. Macerate was plated onto nutrient agar (NA) and incubated at 28°C, yielding colonies that were clear to white in color, smooth, circular and mucoid on NA plates for five suspect isolates (JGD231-235). Strains produced blue diffusible pigment on King's B (KB) media, were Gram-negative rods, and exhibited swimming motility on semi-solid (0.5% agar) NA plates. Crown stab inoculation of ten papaya plants (cv. Red Lady) with isolates resulted in mushy canker symptoms within seven days, while negative control plants stabbed with a sterile probe remained asymptomatic. Koch's postulates were fulfilled by drench-inoculating spontaneous rifampicin-resistant (100µg/ml) mutants, JGD233r and JGD235r, onto ten papaya plants (cv. Solo Sunrise). Nine days post-inoculation, bacterial strains were recovered from symptomatic stem tissue macerate plated on rifampicin (100µg/ml) NA and incubated at 28°C. No symptoms or bacterial cells were recovered from the tissue of negative control plants. Cell morphology, culture phenotypes, and disease symptoms suggested the isolates were Erwinia spp., and blue pigment production on KB further suggested E. papayae (Gardan et al. 2004). Partial 16S rDNA sequences of Guam strains JGD231-235 (sequenced using PCR forward primer 5' - AGAGTTTGATCMTGGCTCAG - 3' and reverse primer 5' - GGTTACCTTGTTACGACTT - 3', GENEWIZ (South Plainfield, NJ)) were deposited into GenBank (OR577627- 631). Highest NCBI BLAST results for all strains showed a 16S rDNA sequence identity of 98.17-98.91% with those of Erwinia sp. I-leaf (LC590218) and E. mallotivora BT-MARDI (HQ456230). A maximum likelihood phylogenetic tree based on concatenated partial atpD, infB, and rpoB sequences of strains JGD232 (PP669340, PP669346, PP669343), JGD233 (PP669341, PP669347, PP669344), and JGD235 (PP669342, PP669348, PP669345) (Brady et al. 2008) constructed using MEGA11 (Tamura et al. 2021) showed all strains formed a monophyletic group with Erwinia sp. I-leaf (Hanagasaki et al. 2020) and E. papayae NCPPB 4294T (Gardan et al. 2004), supported with 98% bootstrap. This note documents the first occurrence of E. papayae as a papaya pathogen in Guam. Papaya cultivation supports sustainable food security for Guam (Bevacqua and Sayama 2023), and Erwinia spp. pathogens threaten papaya on other Pacific islands like Hawaii. These findings convey the need for effective quarantine practices, local disease management, and further research on this pathogen.

First report of Phytophthora niederhauserii causing root rot of carob tree (Ceratonia siliqua L.) in Spain.

Carob tree (Ceratonia siliqua L.) is a crop native to the Mediterranean Basin mainly being used as livestock feed, in pharmaceutical industry, and also a valuable source of human food due to the high dietary fibre and sugar contents. In January 2023, one-year-old 'Duraio' carob trees grafted on 'Rojal' rootstock on pots showing dark brown to necrotic lesions on the secondary roots, decline symptoms and leading to the death of some plants, were detected in a nursery located at the Valencian Community region in Spain. Disease severity was 40 to 50% of root area and disease incidence was approximately 50% on approximately 1000 plants. Six representative plants were randomly collected. Roots were washed under running tap water to rinse the soil away, and small symptomatic pieces were cut, disinfected with 70% ethanol, and then dried on absorbent paper. Two-mm-long segments were plated on CMA-PARBPH, a Phytophthora-selective medium, and incubated at 25ºC. After 2-3 days, growing colonies were transferred to potato dextrose agar (PDA) medium. A Phytophthora-like organism was consistently isolated (50% of root segments, n= 120). Colonies were whitish with irregular margins, had coenocityc mycelium, irregularly branched hyphal swellings, and chlamydospores were absent. Sporangia were non-papillate, persistent, ellipsoid and measuring 25 to 40 × 50 to 90 µm (average: 35.5 × 74.7 µm, n = 50). They proliferated internally with both nested and extended proliferation. These morphological features were similar to those of Phytophthora niederhauserii (Abad et al., 2014). Internal transcribed spacer (ITS) and cytochrome c oxidase I (COX1) regions of a representative isolate CF3 were sequenced to confirm the identity. Both sequences were deposited in GenBank under accession numbers OR763816 for ITS and OR783697 for COX1. OR763816 showed 99.87% sequence identity to P. niederhauserii strain Ex-type MG865552 and OR783697 showed 99.85% sequence identity to P. niederhauserii strain Ex-type MH136944 (Abad et al., 2023). Pathogenicity test was performed on one-year-old carob tree seedlings (Duraio/Rojal) grown in 13 cm-diameter pots to fulfill Koch's postulates. The inoculum was prepared in 1 L glass flasks with a mixture of 200 ml vermiculite, 20 ml oat grains, and 175 ml of V8 broth (20% V8 juice and 0.2% of CaCO3 in demineralized water) (Jung et al., 1996). Glass flasks with vermiculite/oat/V8 mixture were autoclaved three times for 20 min at 120 ºC. These mixtures were inoculated with the isolate CF3 which was previously grown on V8 agar medium and incubated for six weeks in the dark at room temperature (Pérez-Sierra et al., 2013). For inoculation, twenty-gram inoculum were mixed with 200 g autoclaved potting mix (peat, vermiculite and sand; 1:1:1, v/v/v), and added to the pots to plant the seedlings. Seven plants were inoculated and non-infested vermiculite/oat/V8 mixture was used to prepare seven control plants. Two months after inoculation one of the inoculated plants died. Six months after inoculation, only the inoculated plants showed decline symptoms with dry leaves and root necrosis. Isolates resembling P. niederhauserii were recovered by plating the roots from all inoculated plants on CMA-PARBPH, and the identity of the isolates was confirmed as P. niederhauserii based on ITS and COX1 sequencing. To our knowledge, this is the first report of P. niederhauserii causing root rot on carob tree. The detection of this pathogen in nurseries is relevant because its dissemination to orchards could have a negative impact in carob crop production.

Occurrence of grapevine yellow speckle viroid 2 and Australian grapevine viroid in Idaho grapevines.

Grapevine yellow speckle viroid 2 (GYSVd-2; Pospiviroidae, Apscaviroid) causes yellow speckle disease in grapevine (Koltunow et al. 1989) and was found in Australia, Iran, Italy, China, and Nigeria (Koltunow et al. 1989; Habili 2017; Zongoma et al. 2018). In the U.S., GYSVd-2 was found in the State of Washington (Vitis vinifera L. cv. Merlot; Alabi et al., 2012). Australian grapevine viroid (AGVd; Pospiviroidae, Apscaviroid) was reported in Australia, Italy, China, Tunisia, Iran, and in the U.S. wine grapes (V. vinifera) (Habili 2017). In the U.S., AGVd was reported from California (Al Rwahnih et al. 2009), from Washington State (V. vinifera cv. Syrah; GU327604), and from the State of New York (an unknown cv. of V. vinifera; KY081960). In Idaho, two other viroids, hop stunt viroid (HSVd; Pospiviroidae, Hostuviroid) and grapevine yellow speckle viroid 1 (GYSVd-1; Pospiviroidae, Apscaviroid), common in grapevines were previously found in wine grapes (Thompson et al. 2019) but neither GYSVd-2 nor AGVd were identified in the same high-throughput sequencing (HTS) outputs. In September 2020, 16 leaf and petiole samples were collected from six vineyards in Canyon and Nez Perce counties of Idaho, representing six different wine grape cultivars and an unknown table grape cultivar, and subjected to HTS analysis. One of the samples was from a table grape plant at the edge of a declining 'Chardonnay' wine grape block that was grown next to a wine tasting room deck for aesthetic, ornamental purposes; the table grape and 'Chardonnay' plants were own-rooted and planted in 1981. Ribodepleted total RNAs prepared from these samples, as described previously, were subjected to a HTS analysis on a NovaSeq platform (Dahan et al. 2023), producing 15,095,042 to 31,500,611 250-bp paired-end reads per sample. Raw reads were adapter and quality cleaned and mapped against the V. vinifera, reference genome. Unmapped paired-end reads were assembled, and contigs were analyzed using BLASTn and DIAMOND (Buchfink et al. 2021) programs. Fifteen samples were found infected with HSVd and with GYSVd-1, while one was infected with GYSVd-2 and AGVd; in particular, the table grape plant (arbitrarily designated RBTG) was found infected with all four viroid species. The HTS-derived, 490-nt GYSVd-2-specific contig from the table grape sample represented ∼1.35 genome of the Idaho isolate of GYSVd-2 (GYSVd-2-RBTG) and was 100% identical to the GYSVd-2 sequence JQ686716 from Iran. The HTS-derived, 488-nt AGVd-specific contig represented ∼1.32 genome of the Idaho isolate of AGVd (AGVd-RBTG) and was 100% identical to the AGVd sequence KF876037 from Iran. To validate the HTS data and confirm the presence of the four viroids in the original 16 samples, all of them were subjected to RT-PCR using the viroid-specific primers described by Gambino et al. (2014); all 16 samples were found positive for HSVd and GYSVd-1, and one found positive for AGVd. The RBTG sample was confirmed to be infected with HSVd, GYSVd-1, and AGVd by RT-PCR. GYSVd-2 sequence was not amplified, although primers designed by Gambino et al. (2014) matched the HTS-derived GYSVd-2-RBTG sequence; this may be related to a lower concentration of this viroid in the sample and to properties of the primers. The sampled table grape plant was asymptomatic; all four viroids were apparently not associated with any visible abnormalities in this table grape plant, consistent with the findings that viroids found in grapevines typically do not seem to be associated with visible diseases (Habili 2017).

First Report of Penicillium oxalicum Causing Leaf Blight on 'Hongyang' Kiwifruit in China.

In September 2022, leaf blight symptoms (Fig. 1) were detected on six-year-old kiwi trees (Actinidia chinensis cv. 'Hongyang') in Xuzhou municipality (117.29º E, 34.23º N), Jiangsu Province. Early-stage disease symptoms included light brown necrotic lesions of irregular shape ranging in length from 0.2 to 2.4 cm, which turned into leaf blight after approximately 2 weeks. Those symptoms were similar to those previously reported during a Pestalotiopsis sp. infection on kiwi trees in Turkey (Karakaya 2001). Approximately 20% of the leaves from 300 trees examined in one kiwi orchard, 3000 m2 in size, showed the disease symptoms. Ten leading edges of symptomatic leaves were sterilized with 2% sodium hypochlorite for 1 min, rinsed twice with sterile ddH2O and cultured at 26ºC for 3 days on PDA medium containing 50 µg/ml chloramphenicol. The fungal colonies were collected, and the single spore isolation method was used to obtain four isolates. The obtained isolates showed white aerial mycelia that turned greyish after 2 days of cultivation on PDA medium at 26ºC. ITS (OR054113, OR054153-OR054155), TUB2 (OR060951-OR060953, OR249978), and CMD (OR255947-OR255950) genes were amplified using the ITS1/ITS4, BT2a/BT2b and CMD5/CMD6 primers, respectively (Visagie et al. 2014a). The obtained ITS, TUB2, and CMD sequences shared 99.81%-100%, 96.72%-96.96%, and 90.17%-92.58% homology compared to the ex-type strain P. oxalicum CBS 219.30 (MH855125, KF296462, and KF296367), while the obtained ITS and TUB2 sequences showed 99.62%-99.81%, and 96.46%-96.72% identity compared to the representative strain P. oxalicum DTO 179B9 (KJ775647 and KJ775140) (Visagie et al. 2014b). The sequences obtained also showed high homology compared to P. oxalicum HP7-1 (ITS: 99.81%-100% homology; TUB2: 98.98%-99.38% homology; CMD: 94.71%-95.10% homology) (Li et al. 2022). A molecular phylogenetic tree was constructed using MEGA X with representative Penicillium strains retrieved from GenBank (Fig. 2). Microscope observations revealed the presence of curved septate hyphae. Conidia were colorless, unicellular, and ellipsoidal (5-8 µm in length; > 2000 observations), whereas conidiophores were mainly monoverticillate (approximately 20% of the conidiophores were biverticillate) (50-70 µm in length; 43 observations) and contained cylindrical phialides (13-15 µm in length). These findings are consistent with P. oxalicum morphology (Wu et al. 2022; Zheng et al. 2023). The pathogenicity of the four isolates was screened using healthy non-detached 'Hongyang' kiwi leaves. Fifteen leaves from five different two-month-old trees were used for each isolate, with three repetitions. For inoculation, a 10 mL solution containing 1 × 106 spores/mL was sprayed on the leaves. Sterilized water was used in the control experiment, which was carried out using fifteen leaves from five different two-month-old trees, with three repetitions. Inoculated trees were stored at 26ºC and 60% relative humidity for 2 days. All the infected leaves had necrotic lesions and leaf blight symptoms comparable to those found in the field, but the control leaves had no lesions. The pathogen was recovered, and its identity was confirmed by ITS sequencing and morphology analysis, fulfilling Koch's postulates. P. oxalicum is a common cause of blue mould in postharvest fruits (Tang et al. 2020). P. oxalicum has been recently reported as the causal agent of leaf spot in pineapple (Wu et al. 2022; Zheng et al. 2023), and leaf blight on maize (Han et al. 2023). Although Alternaria sp., Glomerella cingulate, Pestalotiopsis sp., Phomopsis sp., and Phoma sp. were previously isolated from kiwi leaves with blight symptoms (Kim et al. 2017), this is the first report of P. oxalicum causing leaf blight on kiwi trees worldwide. P. oxalicum is a well-known source of mycotoxins, such as secalonic acid (Otero et al. 2020), indicating that its presence in kiwifruit orchards may pose a significant risk to human health. The discovery of this hazardous pathogen in kiwi trees must drive the development of management strategies. Kiwifruit is an important dietary source of vitamins, fiber, folate, and potassium, and China is the major producer of kiwifruit, with more than 1.2 million metric tons harvested in 2021. This report will help to generate a better understanding of the pathogens affecting kiwifruit orchards in China.

First Report of Lasiodiplodia pseudotheobromae Causing Necrosis and Canker Disease on Pear Tree in China.

Pear is popular among people, which is an important pillar industry in China. In March of 2023, dark brown necrotic lesions were discovered on the trunks of Pyrus pyrofolia cv. Osmanthus pear in orchard, Liuzhou City, Guangxi Zhuang Autonomous Region. In August, field investigation and sample collection were conducted in orchard. Forty pear trees were selected for symptomatic observation, which of 21 had lesions ranging from 10 to 24 per tree, and 19 with 1 to 8 lesions, respectively. To isolate the pathogen, small tissue pieces of 3 diseased pear trunk samples were disinfected with 75% ethanol for 1 minute, rinsed with sterile water, and dried with filter paper. The tissue pieces were placed on potato dextrose agar (PDA) plates and cultured in a dark incubator at 25℃. Six isolates with the similar morphology were obtained. One of the six isolates was randomly selected as the representative strain and named as GX-3. Mycelium grows with an average rate of 4.26 cm/d. The hypha is highly aerial, and is initially white and then turns black. Subsequently, pycnidia formed and secreted black mucus on the PDA medium after 28 days. The immature conidia were ellipsoid, colorless, hyaline, and unicellular, mostly becoming brown bicellular with longitudinal stripes at maturity. The conidial size was 22.5 to 32.6×12.1 to 19.7µm, and the average size was 28.4±2.3×16.7±2.0 µm (n=50), respectively. GX-3 colony morphology was consistent with that of Lasiodiplodia pseudotheobromae (Alves et al.2008). For molecular identification, the internal transcribed spacer of rDNA (ITS), translation elongation factor 1-α (TEF1-α), and ß-tubulin regions were amplified using the primers ITS1/4, EF1-728F/986R, and Bt2a/Bt2b, respectively (White et al.1990; Carbone and Kohn 1999; Glass and Donaldson 1995). The obtained sequences of GX-3 were deposited in NCBI with Accession numbers OR655421, OR661231, and OR661230, respectively. The sequences of ITS, TEF1-α, and ß-tubulin from GX-3 are 99.44%、99.67% and 99.78% identities with those of L. pseudotheobromae CBS 447.62, respectively. The phylogenetic analysis was performed by maximum likelihood method, revealing that GX-3 is closely clustered with the isolates of L. pseudotheobromae. Therefore, the GX-3 strain was identified as L. pseudotheobromae. GX-3 was further analyzed for its pathogenicity on pear. Firstly, the GX-3 mycelium plugs and spraying spore suspension with the concentration of 1×107 conidia/ml were applied on the stems of 4-month-old healthy birch-leaf pear (Pyrus betulifolia Bunge) potted seedlings by acupuncture needle method, meanwhile PDA and sterile water were used as controls. After 3 days of inoculation, stem surface of the birch-leaf pear exhibited dark brown lesions with slight surface depression, obvious dryness, and canker symptoms, while the control treatment showed no symptoms. The GX-3 was also inoculated on in vitro branches of 'Hosui', 'Hongxiangsu', 'Bodoqing' and 'Xuehua', showing dark brown canker lesions. The same pathogen can be successfully isolated from diseased stems and branches but not from the controls, which accomplishes Koch's postulates. L. pseudotheobromae has been widely reported that it can cause rot and canker on apple, walnut, hackberry, and so on (Xue et al. 2019; Wang et al. 2023; Liang et al. 2020). This is the first report of necrosis and canker disease caused by L. pseudotheobromae on pear in China, which is a potential threat to pear industry.

First report of root and crown rot caused by Fusarium oxysporum Schltdl. on Prunus cerasifera L. in Spain.

In 2021, Spain was the largest producer of cherries in Europe with a production of 125810 tons (FAOSTAT, 2021). In May 2022, in several production fields in Huelva (Spain), wilting was noted in 4-year-old cherry trees cv. Crystal Champaign grafted on rootstock cv. Adara (Prunus cerasifera L.). Gumming and wilting affected approx. 1% of the production area, leading to the eventual collapse and death of most affected trees within 2-3 years. Discoloration of the vascular system of the crown and roots was also noted. Symptomatic crown and root pieces (0.5 cm) were subjected to surface sterilization: immersed in 1% NaClO for 2 min, rinsed in sterile distilled water, and air-dried in a laminar flow cabinet. Then, plant tissues were placed on potato dextrose agar (PDA) amended with streptomycin and incubated in a lab bench at room temperature for a week. Cottony and pink colonies were observed growing from the tissues. Two single strains (F175 and F176) were obtained from each tree by excising single spores (Gordon and Okamoto 1991). Isolates produced sparse aerial mycelia with white to pinkish-orange pigmentation on Spezieller Nährstoffarmer Agar (SNA). Both isolates produced microconidia in false heads on short monophialides. Microconidia were hyaline and measured in the range of 5.0-17.5 × 2.5-3.8 µm for both isolates (n = 50). Macroconidia were less abundant, falciform, and hyaline. Morphological characteristics were consistent with identification as Fusarium spp. (Leslie and Summerell 2006). A portion of the translation elongation factor-1 alpha (EF-1α) gene was sequenced using EF1/2 primers (O'Donnell et al. 1998) (GenBank Accession Nos. OR733348 and OR733349). Based on a comparison of 619 base pairs (bp), both isolates exhibited different sequences, with a 99.5% similarity (616/619 bp). A comparison with previously described isolates revealed a 100% match with published F. oxysporum sequences in the GenBank database (KT323846 and MZ404079, respectively). Isolates were used to conduct pathogenicity tests on 1-year-old plants cv. Adara growing in 512 cm3 pots. Using a scalpel, a 6-7 mm-length wound (2-3 mm deep) was made 5 cm above the soil line in all plants. For each isolate, 5 plants were inoculated by placing a 5 mm plug containing 10-day-old mycelia grown in AMAP medium (Borrero et al. 2009) at the incision point. Non-colonized AMAP plugs were used to inoculate 5 control plants. The inoculated sites were sealed with parafilm. Plants were randomly placed in a growth chamber with a temperature of 28/24ºC and a 12-hour photoperiod. A reddish-brown vascular stem discoloration was noticed in all the inoculated plants 73 days after inoculation. On average, the length of the necrotic area was 12.73 cm for F175, 20.12 cm for F176, and 4.59 cm for the control plants. Fusarium oxysporum was successfully re-isolated from all the inoculated plants. Recovered isolates were confirmed to be the same as the inoculated ones by sequencing the EF-1α gene. A one-way ANOVA indicates that plants cv. Adara were susceptible to both F. oxysporum isolates (P < 0.05). This is particularly noteworthy as cherries are predominantly planted on rootstocks and cv. Adara is a widely used rootstock in Spain. While F. oxysporum has been reported as the cause of root and crown rot in sweet cherry (P. avium L.) in British Columbia (Úrbez-Torres et al. 2016), this is the first report of F. oxysporum causing root and crown rot in cherry rootstocks (P. cerasifera L.) in Spain.

First report of Gymnotelium blasdaleanum causing Saskatoon serviceberry rust in Canada.

Saskatoon serviceberry (Amelanchier alnifolia) is a tall shrub native to the western regions of North America. In British Columbia, serviceberries are a resource for Indigenous communities, used in cooking and for medicinal purposes (Turner et al. 1990). The Saskatoon serviceberry is popular for ornamental and food purposes, with increasing demand for commercial production in British Columbia. (FLNR, 2003). In June 2023 leaf and fruit samples displaying signs of a rust fungus were collected from three ornamental Saskatoon serviceberry shrubs on the University of British Columbia's campus in Vancouver, BC (49°15'35.0"N 123°15'05.6"W). Morphological characteristics were taken from six leaves and berries. Infected leaf tissue was misshapen due to the development of aecia on the abaxial side of the leaves. Approximately 80% of the berries were enveloped in aecia, while infected leaf tissue constituted only around 5% across all three shrubs. Aecia were aecidioid (aecidium type) 0.25 to 0.35mm in diameter, hypophylous and, fructicolous. Peridial cells had a rhomboid shape measuring 21-29 × 18-23 µm (average dimension 25 × 20 µm, n = 30). Aeciospores were globoid with yellowish walls, verrucose and the dimensions ranged from 16-24 × 12-15 µm, (average dimensions 19 ×15 µm, n = 30) (Fig. x). These morphological features fit well with description of Gymnotelium blasdaleanum (Dietel & Holw.) Arthur (= Gymnosporangium libocedri (Henn.) F. Kern) (Arthur 1934). Judging from U.S. National Fungal Databases (https://fungi.ars.usda.gov/) G. blasdaleanum has never been reported in Canada, thus this discovery represents the first report of G. blasdaleanum in the country, specifically in the province of British Columbia. G. blasdaleanum is a hetero-demicyclic rust fungus with a broad aecial host range which includes several Rosaceous genera such as Amelanchier, Crataegus, Cydonia, Malus, Pyrus, and Sorbus (Farr & Rossman 2019). The telial host range is limited to California incense cedar (Calocedrus decurrens) and was not observed on nearby trees, some located within one kilometer of the serviceberries. To confirm morphological identification, DNA was extracted from infected leaves and fruits from all three plants using a modified protocol (Russell et al., 2010). The D1/D2 region was amplified using primers LR6 and 2Rust1R (Beenken, et al., 2012). A BLASTn search of all three sequences revealed 99.52 to 99.62% identity to available sequence in GenBank from Gymnotelium blasdaleanum (AF522168). Newly generated sequences were submitted to the GenBank under accession numbers OR567878, OR568568, and OR654105. Specimens from three Saskatoon serviceberry plants were provided to the UBC herbarium (Beaty Biodiversity Museum) and deposited in their fungarium (F35820). Infected fruits undergo deformation and premature dropping, posing a significant threat to ornamental and fruit production varieties of serviceberries (EPPO, 2006). The spread and establishment of the G. blasdaleanum is highly dependent on the presence of C. decurrens because of its limited range. However, California incense cedar is planted outside its natural range in suitable environments for G. blasdaleanum to thrive such as southern BC allowing the rust pathogen to spread to Canada. To prevent potential spread into other agricultural industries, particularly the 256-acre pear cultivation, continued planting of C. decurrens in British Columbia should be closely monitored. (BC MFLNRO, 2003).

A new method in the production of protective sheets against X-ray radiation.

Today, the use of X-rays in diagnosing and sometimes treating patients is inevitable. Despite the many benefits of using X-rays in medical and other sciences, the harmful effects of this radiation on human tissue should not be neglected. One of the best ways to prevent the harmful effects of X-rays on the human body is to use appropriate covers against these rays. It seems that it is necessary to find effective particles to weaken X-rays and choose a suitable substrate with high mechanical resistance to scatter particles in it. In this study, the synthesis of SnO2 nanoparticles from SnCl2.2H2O precursor and BaSO4 nanoparticles from BaCl2.2H2O precursor using neem tree extract (Azadirachta indica) as a reducing and stabilizing agent is reported. After the synthesis of nanoparticles, their structure was investigated by X-ray diffraction, scanning electron microscopy and energy dispersive X-ray analysis. Then the desired composite and nanocomposite were prepared in the polymer substrate. The sheets were prepared using an extruder and then a hot hydraulic press. The output sheets had a thickness of 1 mm. The structural characteristics of the produced sheets such as surface morphology, density of prepared composites, mechanical properties, thermal gravimetric analysis and retention of loaded particles after three times washes were investigated. The X-ray attenuation capability of each sample was evaluated by calculating the linear attenuation coefficient for each prepared sample. The results show that all sheets filled with tin and barium micro and nano particles have more X-ray attenuation capabilities than pure polymer. Among the prepared sheets, the nanocomposite prepared from low-density polyethylene (77 %) + SnO2 (10 %) + BaSO4 (10 %) + multi-walled carbon nanotubes (3 %) showed the highest X-ray attenuation.

Survey on Latent Infection of Canker-Causing Pathogens in Budwood and Young Trees from Almond and Prune Nurseries in California.

Almond band canker and prune Cytospora canker have become more severe in the last decade, especially in young orchards, in California. To test our hypothesis that young trees from nurseries could carry the canker-causing pathogens at latency phase to new orchards through transplanting, a multiyear survey on latent infection of canker-causing pathogens of budwood and young trees of almond and prune nurseries in California was conducted. A total of more than 1,730 samples including shoots of rootstocks and scions and grafting union cuttings were collected from 11 nurseries. A real-time quantitative PCR assay was applied to quantify the latent infection levels by six canker-causing pathogen taxa: Botryosphaeria dothidea and species of Cytospora, Diplodia, Lasiodiplodia, Neofusicoccum, and Phomopsis. For almond, the average incidences of latent infection caused by Lasiodiplodia spp. (43.6%) and Neofusicoccum spp. (24.2%) were significantly greater than those by the other four pathogen taxa. The molecular severity (MS) of latent infection caused by Neofusicoccum spp. (3.6) was significantly greater than those caused by other pathogen taxa, except for Lasiodiplodia spp. (2.6). For prune, the average incidence of latent infection caused by Cytospora spp. (13.5%) was significantly higher than those caused by B. dothidea (1.5%) and Diplodia spp. (1.3%) but not significantly higher than those caused by Lasiodiplodia spp. (6.9%), Neofusicoccum spp. (6.3%), and Phomopsis spp. (7.7%), respectively. Moreover, the average MS values caused by Cytospora spp. (3.8) and Neofusicoccum spp. (3.2) were the highest followed by those caused by B. dothidea (1.4), Lasiodiplodia spp. (2.2), and Phomopsis spp. (2.3). Different almond varieties showed various levels of susceptibilities to different canker-causing pathogens. This study concluded that Lasiodiplodia and Neofusicoccum are the predominant pathogen species in almond, and Cytospora is the most important canker-causing pathogen species in prune in nurseries. These findings confirmed the observations of predominancy of canker-causing pathogens in almond and prune orchards in California.

Reassessing the Etiology of Aspergillus Vine Canker and Summer Bunch Rot of Table Grapes in California.

Fungal taxonomy is in constant flux, and the advent of reliable DNA barcodes has enabled the enhancement of plant pathogen identification accuracy. In California, Aspergillus vine canker (AVC) and summer bunch rot (SBR) are economically important diseases that affect the wood and fruit of grapevines, respectively, and their causal agents are primarily species of black aspergilli (Aspergillus section Nigri). During the last decade, the taxonomy of this fungal group has been rearranged several times using morphological, physiological, and genetic analyses, which resulted in the incorporation of multiple cryptic species that are difficult to distinguish. Therefore, in this study, we aimed to reassess the etiology of AVC and SBR using a combination of morphological observations with phylogenetic reconstructions based on nucleotide sequences of the calmodulin (CaM) gene. Results revealed that the isolates causing AVC from recent isolations corresponded to A. tubingensis, whereas the isolates obtained from initial surveys when the disease was discovered were confirmed as A. niger and A. carbonarius. Similarly, the isolates obtained from table grapes with SBR symptoms and from spore traps placed in those vineyards were identified primarily as A. tubingensis, followed by A. niger and A. carbonarius. Notably, the A. niger isolates formed a subclade with strains previously known as A. welwitschiae, which is a species that was recently synonymized with A. niger. Overall, the most prevalent species was A. tubingensis, which was associated with both AVC and SBR, and representative isolates recovered from AVC-symptomatic wood, berries SBR symptoms, and spore traps were equally pathogenic in healthy wood and berries of 'Red Globe' grapevines. This study also constitutes the first report of A. tubingensis causing AVC and SBR of grapes in California and in the United States.

First report of three bunya-like viruses, apple luteovirus 1, and apple hammerhead viroid in apples from Hakkari, Türkiye.

Türkiye is a major apple fruit producer in the crossroads of Europe and the Middle East. Several reports have described the presence of multiple viruses affecting apple production in Türkiye, including apple stem grooving virus (ASGV), apple stem pitting virus (ASPV), apple chlorotic leafspot virus (ACLSV), and apple mosaic virus (ApMV) (Kurçman 1977; Fidan 1994; Çaglayan et al. 2003). However, there are no reports of the presence of the recently discovered bunya-like viruses citrus concave gum-associated virus (CCGaV), and apple rubbery wood viruses 1 and 2 (ARWV1 and 2), as well as apple luteovirus 1 (ALV-1), and apple hammerhead viroid (AHVd) in Türkiye, all of which have been previously reported in other apple-producing countries (Wright et al. 2018; Liu et al. 2018; Zhang et al. 2014). Leaves from one Gala, two Granny Smith, and one Golden Delicious apple trees showing mild symptoms of curling, chlorosis, and yellowing were collected from four different orchards in the province of Hakkari, southeast Türkiye during June 2022 and sent to USDA APHIS Plant Germplasm Quarantine Program (under permit) for virus and viroid HTS-based diagnostics. Total RNA was isolated using the RNeasy Plant Mini Kit (Qiagen) following the manufacturer's guidelines to prepare RNAseq libraries using the TruSeq Stranded Total RNA Library Plant Kit (Illumina, Inc) as described in Malapi-Wight et al. (2021). Libraries were sequenced on the NextSeq500 sequencer (PE 2x75), and approximately 45 million reads were obtained per each sample on average. Bioinformatic analysis was performed as described in Costa et al. (2022) using Phytopipe, where unclassified pathogen-derived reads were de novo assembled and contigs were compared to the NCBI viral nucleotide and protein databases by BlastN and BlastX respectively using a 10-4 e-value cutoff. Nearly complete genome contigs were obtained for ACLSV (OR640150) and ASPV (OR640151) in all four samples and for ASGV (OR640152) in 3 of the 4 samples. The average BlastN identity to sequences in GenBank was 92.3% for ACLSV, ranging from 89-94 %. BlastN identity for ASPV was 86%, ranging from 81-92 % while the ASGV average BlastN identity was 98.2%. Nearly complete genomes with average genome coverage of 92.4% and 95.6% for RNA1 and RNA2 of CCGaV (OR640153 and OR640154), were found in two of the four samples with BlastN identity of 94.7% and 94.8% to GenBank sequences. Additionally, nearly complete genome of the large (L), medium (M), and small (S) segments for ARWV1 were found in two samples with average genome coverage of 99.9%, 99.4%, and 100% respectively and BlastN identity of 98.8%, 95.2%, and 98.4% (OR640155, OR640156, OR640157). ARWV2 contigs were also found in 1 sample where M and S segments had a coverage of 99.8% and BlastN identity of 95.4% (OR640158 and OR640159). The nearly complete genome of ALV-1 was also found in two of four samples with genome coverage of 94.1% and an average BlastN identity of 93.4% (OR640160). AHVd was found in one of the Granny Smith trees with 19,260 mapped reads to the reference GenBank MH049335.1 and identity of 98.3% (OR640149). The HTS findings of CCGaV, ARWV1, ARWV2, and ALV-1, from Türkiye were later confirmed by Sanger sequencing using custom-designed primers targeting the coat protein, the RNA-dependent RNA polymerase, or ~390bp for the AHVd genome (Supplementary Table 1). To further learn about the incidence of these agents, we tested 12 other apple samples from six different neighboring orchards and found them at 18.8% rate for CCGaV, 12.5% for both ARWV1 and ARWV2, 25% for ALV-1, and 37.5% for AHVd respectively. To our knowledge, this is the first report of the apple viruses CCGaV, ARWV1, ARWV2, and ALV-1, and the AHVd viroid in Türkiye. Further studies of the impact of these agents on orchard's health are necessary, including their prevalence in high apple production regions of Türkiye.

First Report of Fusarium luffae Causing Leaf Blight on Loquat in Eastern China.

During May-June 2021 and 2022, leaf blight symptoms were observed on loquat leaves (Eriobotrya japonica cv. 'Mogi') in Jiangsu Province (Xuzhou municipality, 117.17° E, 34.13° N) in China. Approximately 10% of the leaves on the two hundred trees studied in a six-year-old loquat orchard exhibited round lesions that changed from light yellow to reddish-brown in 8-10 days. Approximately 3% of the infected leaves exhibited numerous lesions that coalesced, leading to expansive blighted areas. Twenty-five samples of symptomatic tissue, approximately 0.2 cm2 in size, were collected in May 2022 from five different trees (five samples per tree), sterilized in 2% NaOCl for 1 min, washed twice with sterilized ddH2O, and incubated at 26°C for 5 days on PDA medium containing 50 µg/mL chloramphenicol. Six isolates were obtained via single spore isolation. ITS (OQ954852-OQ954857), TUB2 (OQ968488-OQ968493), EF1-α (OQ971890-OQ971895), RPB1 (OQ971896-OQ971901), and RPB2 (OR037266-OR037271) genes were amplified using the ITS1/ITS4, T1/T22, EF1-728F/EF1-986R, RPB1-R8/RPB1-F5, and fRPB2-7CF/fRPB2-11aR primers, respectively (O'Donnell et al. 2010). The species was identified using the Fusarioid ID database (Crous et al. 2021), revealing that all obtained isolates showed high homology to representative F. luffae strains. Upon combining the ITS, TUB2, EF1-α, RPB1, and RPB2 sequences, the isolates showed 99.42%-97.85% and 99.59%-98.10% identity to F. luffae CGMCC 3.19497 (ex-type strain) and NRRL 32522, respectively. A molecular phylogenetic tree was constructed using MEGA X, with a selection of representative Fusarium strains. Microscope observations showed septate mycelium, microconidia (6.86 ± 0.91 µm length, 1.67 ± 0.24 µm width, containing 1 septum; number of observations = 21), fusiform macroconidia (15.88 ± 1.43 µm length, 1.66 ± 0.24 µm width, containing 1 septum; number of observations = 45), and linear chlamydospores (79.36 ± 28.36 µm length, 12.03 ± 3.37 µm width; number of observations = 152). These observations are consistent with the morphology of F. luffae (Wang et al. 2019). All isolates exhibited identical morphological characteristics. All isolates were evaluated for pathogenicity in vivo using healthy non-detached loquat leaves. A total of 15 leaves from 5 different three-month-old 'Mogi' loquat trees were used for each isolate. Experiments were performed three times. A suspension of 1 × 106 spores/mL obtained from a seven-day-old colony (10 mL per 15 leaves), was sprayed on non-wounded leaves for inoculation. Sterilized ddH2O was used in the control experiment. Inoculated trees were stored at 26°C and 70% relative humidity for four days. Leaf blight symptoms were observed in all inoculated leaves, and the symptoms were observed in all repeated trials. The pathogen was recovered, and its identity was confirmed by ITS sequencing and morphological analysis, fulfilling Koch's postulates. In recent years, F. luffae has been reported to cause fruit rot on muskmelon, flower rot on kiwifruit, soybean pod rot, and leaf spot on cherry in China (Yu et al. 2022; Zhang et al. 2022; Zhao et al. 2022; Zhou et al. 2022), demonstrating the host promiscuity of this pathogen. Although F. solani has been identified as the causal agent of root rot and fruit rot on loquat (Abbas et al. 2017; Wu et al. 2021), this is the first report of F. luffae causing leaf blight on loquat worldwide. This report will help to understand the pathogens affecting loquat orchards in China.

First report of Irpex rosettiformis causing white root rot in avocado trees in Michoacán, México.

In México, avocado production is an important economic source. In the last season it generated $ 3. 27 billion USD of foreign currency in the country. Irpex spp. are wood decay fungi. In the period 2019-2022, in the state of Michoacán (19°13' N; 101°55' W), México, basidiomes of Irpex sp. were observed on the base of trunks and crowns of 5-years-old and older avocado (Persea americana) trees. The trees exhibited disease symptoms that included white root rot, leaf yellowing, small leaves, branch diebacks, generalized defoliation, apical flaccidity, abundant but small sun burnt fruits due to the lack of foliage, and after 2-4 years of first disease appearance, the infected trees died. In the place where fungus was established, abundant white and cottony mycelium was formed, which caused trees decay. The incidence of the disease in the sampled orchards was estimated to be 30% per ha with 350 - 400 trees, which was determined through a simple sampling design focused on trees with signs and symptoms of the disease due to the phytopathogen. Samples of infected tissue (roots and stems) and fungal basidiomes were collected from 90 trees (5-6 per orchard). The symptomatic avocado trees studied were randomly selected from 17 orchards. For the fungal macroscopic characterization, the synoptic keys described by Gilbertson and Ryvarden (1986) and by Largent (1973) were used. The samples showed typical structures corresponding to Irpex sp., including rosettes, annual basidiomes, a system of monomitic hyphae, and subglobose basidiospores. In vitro fungal isolation from basidiomes and infected tree tissues was done according to the protocol of Agrios (2004). The fungal strains were maintained on PDA at 28 °C. At 16 days of incubation the colonies were opaque, whitish with fluffy and corky mycelium. Microscopic analysis of the fungus showed typical yellowish spores, with an ellipsoid shape of 3-4 x 4-5.5 µm (50 accounted structures per isolate [N=19]) and basidia of 20-25 x 4.5-5.5 µm (n=20 basidiomes). For molecular characterization, two molecular markers were used, the internal transcribed spacer rDNA-ITS1 5.8 rDNA-ITS2 (ITS; White et al. 1990) and the large ribosomal subunit (LSU; Vilgalys and Hester 1990). The PCR reaction was performed as described by Martínez-González et al. (2017). The consensus sequences were compared with those deposited in the NCBI-GenBank, using the BLASTN 2.2.19 tool (Zhang et al. 2000), the samples showed 99% match with the species, Irpex rosettiformis. GenBank accession numbers of the submitted isolates are summarized in supplementary Table 4. To test Koch's postulates, 3-months old avocado plants grown in greenhouse conditions were inoculated (n = 10 per each isolate [N= 19]) on the roots with 3 g of I. rosettiformis mycelium. The experiment was done twice with 20 non-inoculated plants as control. After 67 days, basidiomes (50 x 70 x 1.5 mm in average) were observed where the disease incidence was >77%, with subsequent tree decline. The pathogen was re-isolated in vitro in PDA and its identity was confirmed by morphological characteristics of mycelium. This work shows that I. rosettiformis is not only a wood decay fungus, but also a phytopathogen, the causative agent of white root rot disease in P. americana var. drymifolia, cultivar 'Hass', which establishes a precedent for monitoring and preventing its proliferation to other regions in the American continent and the world where nursery avocado seedlings are exported.

Occurrence of Neofusicoccum parvum Causing Canker and Branch Dieback of European Hazelnut in Maule Region, Chile.

The European hazelnut (Corylus avellana) is an important fruit crop cultivated in Chile, with over 17,000 ha planted (46%) in the Maule region, central Chile. During a routine orchard survey in seasons 2020-2021 and 2021-2022, in the Maule region, canker and dieback symptoms were observed in two commercial orchards of European hazelnut cv. Tonda Di Giffoni in San Rafael (8-year-olds) and Linares (15-year-olds), with an incidence between 10% and 36%, respectively, based on external symptoms. Twenty symptomatic branches exhibiting cankers, reduced vigor, wilting, twig death, and dieback, were collected. A cross-section of diseased branches revealed mostly brown V or U-shaped cankers of hard consistency. Branches were cut, and pieces of cankers were surface sterilized in 96% ethanol for 3 s and briefly flamed. Small pieces of wood (5 mm2) from the edge of cankered tissues were placed on Potato Dextrose Agar (2% PDA) amended with 0.1% Igepal CO-630 and incubated at 25°C for five days in the dark (Díaz and Latorre 2014). Pure cultures were obtained by transferring a hyphal tip from growing colonies to fresh PDA media. Eight pure cultures (NP-Haz01 to NP-Haz08) developed dark to olive-brown fast-growing colonies with scarce aerial mycelium after seven days at 25°C on PDA under near-UV light. These isolates showed a dark-olive color on the reverse side of Petri dishes and developed abundant, aggregated, and dark-brown globose pycnidia after 21 days at 25°C. Conidia were hyaline, aseptate, ellipsoidal, densely granulate, externally smooth, and thin-walled dark, that measured (9.5-) 15.5 ±1.2 (-17.3) x (5.1-) 7.2 ± 0.6 (-9.1) µm (n = 30), with a length/width ratio of 2.15. These isolates were tentatively identified morphologically as Neofusicoccum sp. Molecular identification was performed using ITS1/ITS4, Bt2a/Bt2b and EF1-728F/EF1-986R primers of the internal transcribed spacer (ITS1-5.8S-ITS2) region, a portion of the beta-tubulin (BT) and part of the translation elongation factor (EF1-) genes, respectively (Dissanayake et al. 2015). A MegaBlast search in GenBank showed a 99% similarity to isolate CMW9081, the ex-type of Neofusicocum parvum (Pennycook & Samuels) Crous, Slippers & A.J.L. Phillips. The sequences were added to GenBank (OR393855 to OR393857 for ITS; OR400688 to OR400690 for BT; OR400691 to OR400693 for EF1-). Pathogenicity of three isolates (NP-Haz02, NP-Haz04, NP-Haz09) was studied on freshly made pruning wounds on attached branches of 3-year-old and one-year-old of European hazelnut cv. Tonda Di Giffoni in the San Rafael field. Fifteen pruning wounds were inoculated with 40 µL conidial suspension (105 conidia/mL) of each isolate of N. parvum. Sterile distilled water was used as a control treatment (n=15 branches) for branches of 3-year-olds and one-year-olds. Both pathogenicity tests were repeated once. Attached branches of 3-year-olds (6 months of incubation) and one-year-olds (4 months of incubation), developed necrotic streaks and cankers with a mean length of 33 to 82 mm and 25 to 51 mm, respectively. No necrotic streaks were observed in the branches treated with water. Neofusicoccum parvum was reisolated only from symptomatic tissues of inoculated branches, and morphological and molecularly (EF1-) identified, thus fulfilling Koch's postulates. Previously, other Botryosphaeriaceae spp. as Diplodia coryli (Guerrero and Pérez 2012) and D. mutila (Moya-Elizondo et al. 2023) have been obtained from canker and dieback of hazelnut in Chile. Recently, N. parvum was reported causing nut rot in hazelnuts in Italy (Wagas et al. 2022). To our knowledge, this is the first report of N. parvum causing canker and branch dieback of hazelnut trees in Chile and worldwide.

First Report of Leaf Spot Disease on Pineapple Caused by Fusarium solani in China.

Pineapple (Ananas comosus) is an economically important tropical fruit in Guangdong Province, China. The plants were seriously infected with a year-round leaf spot disease. During September to November 2022, the leaf spot disease of pineapple was found in Xuwen city and Zhanjiang city of Guangdong Province. A disease survey of 1 ha revealed that pineapple was affected at an incidence ranging from 30% to 50%. The disease caused economic loss to control plant diseases with chemicals. The initial symptoms were observed after 1 month of planting in October and included yellow spot and developed brown necrotic lesions. The leaves of pineapples showed symptoms of Large brown necrotic lesions appear on leaves especially on the tip of basal leaves. The diseased leaves were collected and surface-disinfested in 1% NaClO for 2-3 m, rinsed with sterilized water, air dried, placed on potato dextrose agar (PDA) medium, and incubated for 3 to 5 days at 28°C. Two isolates (PLF1 and PLF2) were collected and purified using the single-spore method. Colonies developing on PDA were in a circle with abundant white, densely fluffy aerial mycelium, pale to colorless after 3 d and pale orange 5-15 d later. Cultures of the isolates produced macroconidia which were falcate, 1-3-septate, hyaline, 18.2-43.4×4.8-6.8 µm(n=50). Cultures of the isolate also produced large amount of conidia which were hyaline, oblong, no septate, 5.2-10.6×2.7-5.2 µm (n=50). These characteristics were consistent with the description of Fusarium sp. (Chitrampalam et al. 2018). For molecular identification, the genomic DNA of the 2 isolates was extracted. The fragments of internal transcribed spacer (ITS), translation elongation factor 1α (EF1α) and ß-tubulin were amplified and sequened using the primer pairs of ITS4/ITS5, EF1/EF2 (O'Donnell et al. 2008) and T1/T2 (O'Donnell et al. 1997). These sequences were deposited in GenBank (OR501466, OR501467 for the ITS; OR499874, OR499875 for elongation factor; OR499876, OR499877 for ß-tubulin). Phylogenetic trees were constructed in MAGA 11 using the Maximum likelihood (ML) method based on the concatenated sequences of ITS, EF1α, and Tublin (Figure 1). The 2 isolates were grouped with F. solani A01-1 (GCA_027945525.1) with a bootstrap value of 100 in the phylogenetic tree. The morphology and multi-gene phylogenetic analysis indicated that the new isolates are F. solani. The 2 isolates were selected for pathogenicity tests to fulfill Koch's postulates. Six plants at seven- to ten-leaf stage were inoculated with each isolate separately. Three sites of each leaf were wounded with a sterile needle and covered with a piece of cotton drenched with 200 µl spore suspension (107 spores/ml) from each isolate cultured in PD medium. Leaves inoculated with PD medium served as negative controls. Inoculated plants were placed in an incubator at 28°C, and 80% humidity under a 12-h light/dark cycle for 7 days. After 7 days of incubation, necrotic spots were observed in all the inoculated plants except the negative control. The pathogenicity tests were conducted three times with similar results. The strains were then reisolated from the lesions and found to be Fusarium solani as those of the inoculum. F．ananatum， F. guttiforme and F. subglutinans have been reported to infect all parts of pineapplefusariosis disease (Jacobs et al. 2010; Stepien et al. 2013; Ventura et al. 1993). To our knowledge, this is the first report of fusariosis on Pineapple caused by F. solani. Identification of F. solani as a disease agent on pineapple will assist in disease management for this important fruit tree.

First report of Diplodia bulgarica causing black canker on apple in California.

California is the sixth largest apple-producing state in the United States with a production that reached 4,654 ha in 2021. During the late winter of 2023, black canker symptoms were observed on branches of 'Gravenstein' apple (Malus domestica) in two commercial orchards in Sonoma County, California. The prevalence of symptomatic trees ranged from 10 to 30%. External symptoms included charcoal looking-cankers with the bark peeling off from the primary and secondary branches. Internally, cankers were dark brown in color with a hard consistency. Pycnidia were observed on the surface of older cankers. Fungal isolations were performed from disinfected (70% ethanol, 30 s) symptomatic branch samples (n = 15). Small wood pieces (5 mm length) were taken from the margin of diseased and healthy tissues, and placed on potato dextrose agar acidified with 92% lactic acid at 0.5 mL per liter (APDA). Plates were incubated at room temperature (20-22 °C) for 7 days. Colonies of Botryosphaeriaceae species (Phillips et al. 2013) (n = 12) were consistently recovered and pure cultures were obtained by transferring a single hyphal tip onto fresh APDA. Colonies were light gray with irregular margins. To induce pycnidia formation, two isolates (UCD11350 and UCD11351) were grown on pistachio leaf agar for 21 days. Conidia (n = 50) were thick-walled and ovoid in shape, initially hyaline, then turned pale brown and dark brown at maturity, and some of them became 1-septate, ranging from 18.9 to 24.0 (21.9) × 11.5 to 14.7 (13.4) µm. Isolates were identified by sequencing a partial region of the beta-tubulin (tub2) gene using the primers Bt2a/Bt2b (Glass and Donaldson 1995). BLAST searches on NCBI GenBank revealed 99.5 % identity with the Diplodia bulgarica ex-type (CBS 1245254). To confirm the identity, the rRNA internal transcribed spacer (ITS) and the translation elongation factor 1-alpha (tef1) were also sequenced using ITS5/ITS4 (White et al. 1990), and EF1-688F/EF1-1251R (Alves et al. 2008), respectively. A maximum parsimony multi-locus phylogenetic analysis clustered Californian isolates with reference strains of D. bulgarica. Sequences were deposited in GenBank (nos. OR631209 to OR631210, OR637361 to OR637362, OR637363 to OR637364 for ITS, tub2, and tef1, respectively). Pathogenicity tests were conducted on 2 to 3-year-old branches (n = 5) of over 20-year-old trees by inserting a 5-mm segment of a toothpick, completely colonized with each of the two isolates mentioned above, into a 1-mm-diameter hole made with a sterile drill bit. The same number of branches where mock inoculated with a non-colonized toothpick as negative control. The experiment was performed twice. After ten weeks, inoculations resulted in dark brown necrotic lesions that ranged from 54.0 to 59.8 mm in length. Negative controls remained asymptomatic. Koch's postulates were fulfilled by successfully recovering the isolates from the lesion margins, which were confirmed by morphology. Diplodia bulgarica was first described affecting M. sylvestris in Bulgaria (Phillips et al. 2012), and then detected on M. domestica causing cankers in Iran (Abdollahzadeh 2015), India (Nabi et al. 2020), Germany (Hinrichs-Berger al. 2021) and Türkiye (Eken 2021). The pathogen was also identified causing postharvest fruit rot (Eken 2022). To our knowledge, this is the first report of D. bulgarica causing branch canker on apple in California, which provides important information for developing detection and control strategies.

Irrigation Targeted to Provoke Ejection of Ascospores of Venturia inaequalis Shortens the Season for Ascospore Release and Results in Less Apple Scab.

Trials were carried out in apple orchards of Emilia-Romagna and Trentino-Alto Adige in northern Italy to investigate the effects of sprinkler irrigation on possible reduction in inoculum and subsequent disease pressure of Venturia inaequalis, the ascomycete causing apple scab. In spring, volumetric spore traps were placed above apple leaf litter containing pseudothecia with ascospores of the fungus. Pseudothecia matured more rapidly in irrigated plots, and 95% of the total number of spores trapped in a season was reached on average 164 degree days (base temperature 0°C) earlier in irrigated compared to non-irrigated plots. On average for seven location/year combinations, more than 50% of the ascospores were trapped following irrigations carried out for two hours on sunny days before a forecasted rainfall. Subsequently, a much lower number of spores were trapped on rainy days following irrigation. Field trials with scab susceptible apple cultivars were carried out in the two regions to evaluate the efficacy of sprinkler irrigation on disease. Irrigated and non-irrigated plots were either treated with different fungicide control strategies or not treated. Irrigation significantly reduced the incidence of apple scab at both sites, and the overall number of infected leaves and fruit was reduced by more than 50%. Mid-day sprinkler irrigation can significantly reduce the inoculum pressure of V. inaequalis in apple orchards. This may be a sustainable management strategy, especially in areas with extended dry periods.

Identification and distribution of the "Candidatus Liberibacter asiaticus"-Asian citrus psyllid pathosystem in Saudi Arabia.

Citrus greening disease was first reported in Saudi Arabia during the 1970's when characteristic foliar and fruit symptoms were observed in commercial citrus groves, however, "Candidatus Liberibacter asiaticus" (CLas) was not detected in symptomatic trees until 1981-1984 when CLas-like cells were observed by transmission electron microscopy in leaves collected from symptomatic citrus groves in southwestern Saudi Arabia. Despite the anticipated establishment of the CLas-Asian citrus psyllid (ACP) (Diaphorina citri Kuwayama) pathosystem, CLas presence has not been verified in suspect trees nor have ACP infestations been documented. Given the recent expansion of citrus production in Saudi Arabia, a systematic country-wide survey was carried out to determine the potential CLas distribution in the thirteen citrus-growing regions of the country. Citrus trees were surveyed for presence of CLas-psyllid vector(s) and characteristic disease symptoms in commercial and urban citrus trees. Adult psyllids collected from infested citrus trees were identified as ACP based on morphological characteristics. Real-time, quantitative PCR amplification (qPCR) of the CLas ß-subunit of the ribonucleotide reductase (RNR) gene from citrus leaf and fruit samples and/or ACP adults, revealed trees were positive for CLas detection in ten of the 13 survey regions, however, CLas was undetectable in ACP adults. Phylogenetic and SNPs analyses of a PCR-amplified, cloned fragment of the CLas 16S rRNA gene (~1.1 kbp) indicated Saudi Arabian isolates were most closely related to Florida, USA isolates. Analysis of climate variables indicated that the distribution of the ACP-CLas pathosystem observed in Saudi Arabia was consistent with published predictions of terrains most likely to support establishment.